Rapid signaling between neurons (and other types of cells) is mediated by ligand-activated ion channels. The best studied examples are the pentameric nicotinic acetylcholine receptor channels and the tetrameric glutamate receptor channels. Our work on ligand-activated channels has focused on P2X receptor channels, a family of cation channels that are activated by extracellular ATP. P2X receptor channels are distinct from classical ligand-activated ion channels in that they are trimers, and the activating ligand can be released synaptically, via large-pore channels, or from dying cells. Our work thus far has focused on the structure, gating mechanisms and pharmacology of P2X receptors. Each subunit of a P2X receptor contains two TM segments, a large intervening extracellular ATP binding domain, and intracellular N- and C-termini. Our earliest studies on P2X receptor channels used scanning mutagenesis to show that the two TMs adopt helical secondary structure, and explored the mechanism by which Ivermectin stabilizes the open state of P2X receptor channels. This interesting lactone is widely used to treat river blindness, and although it is known to interact with a range of ligand-gated ion channels, its site and mechanism of action were unknown. Our studies on the mechanism of IVM facilitation of P2X receptor channels suggests that the hydrophobic lactone partitions into the membrane to bind to TM helices at the protein-lipid interface to stabilize the open state. A recent X-ray structure of an open glutamate-activated chloride channel in complex with Ivermectin shows the lactone bound to TM helices at the protein-lipid interface, supporting our proposed mechanism of action of Ivermectin on P2X receptor channels. We also employed Cys accessibility approaches to show that the TM2 helix lines the pore and that the gate is located within the outer portion of that helix, conclusions that were confirmed by a recent X-ray structure of a P2X receptor in a closed state. The X-ray structure of the P2X receptor reveals many unanticipated features of these trimeric ion channels; the pore-lining TM2 helices are steeply angled with respect to the membrane (50 degrees), and the transmembrane subunit interface is formed exclusively by packing between TM2 helices at the three-fold axis. Our accessibility results suggest that the extended hydrophobic plug formed by the packing between TM2 helices functions as a gate that must expand to allow ions to permeate. Based on engineered metal bridges at the central axis of the internal pore, we propose a model in which the TM2 helices must straighten to open the pore. One key prediction of this new mechanism is that upon straightening, the TM2 helices reposition to form subunit interfaces with the TM1 helices of adjacent subunits. We propose to investigate the structure of the open pore by engineering metal bridges and mapping the subunit interface. One interesting phenomena observed with P2X receptor channels is that upon prolonged activation by ATP, cells exhibit an increased permeability to relatively large organic cations. We propose to investigate whether this dynamic ion selectivity results from a change in the pore of P2X receptors and to explore its structural and mechanistic basis using Cys accessibility and engineered metal bridges.